Biogenic amine transporters play important roles in the action and recycling of their specific neurotransmitter substrates. They have been well established as the targets for many pharmacological agents that affect brain function. For examples, the rewarding properties ofthe abused drugs, cocaine and amphetamine, are due mainly to its inhibition of dopamine transporter (DAT); the antidepressant effect of imipramine is exerted on serotonin transporter (SERT). These transporters belong to the Neurotransmitter:Sodium Symporter (NSS) family, which has a large number of prokaryotic homologs. A prokaryotic leucine transporter (LeuT) structure has been solved in high resolution, with the substrate Leu bound in an enclosed cavity. However, the determinants of substrate specificity, an understanding of which is important for rational drug design, may lie not only within the binding site crevice revealed by the LeuT structure, but also along the permeation pathway. The long term goal of the present proposal is to elucidate dynamic structural changes of the permeation pathway of NSS family proteins during the translocation cycle and to evaluate competing transport models, e. g., the alternating-access scheme and our two-substrate model. Specifically, using prokaryotic NSS-proteins as model systems, this will be achieved by an integrated, comparative process of iteration between molecular modeling simulations and experimental investigations/validations. With this multidisciplinary protocol we will explore the conserved residue positions and potential auxiliary cavities that line the permeation pathway and reorganize dynamically in different conformational states. The common, and the different features of the permeation pathways, including the roles of sodium ions, will be compared among prokaryotic and eukaryotic NSS-proteins. The knowledge acquired should shed light on the translocation cycles of biogenic amine transporters and will contribute to our understanding of the structural bases of substrate specificities.